EP0132178B1 - Process for the preparation of the principal proteins of hemolysed blood in a non denatured form - Google Patents

Process for the preparation of the principal proteins of hemolysed blood in a non denatured form Download PDF

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Publication number
EP0132178B1
EP0132178B1 EP84401303A EP84401303A EP0132178B1 EP 0132178 B1 EP0132178 B1 EP 0132178B1 EP 84401303 A EP84401303 A EP 84401303A EP 84401303 A EP84401303 A EP 84401303A EP 0132178 B1 EP0132178 B1 EP 0132178B1
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Prior art keywords
process according
effected
albumin
chromatography
blood
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German (de)
French (fr)
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EP0132178A1 (en
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J.L. Tayot
Paule Annie Gattel
Michel André Tardy
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Sanofi Pasteur SA
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Institut Merieux SA
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/76—Albumins
    • C07K14/765—Serum albumin, e.g. HSA
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/795—Porphyrin- or corrin-ring-containing peptides
    • C07K14/805—Haemoglobins; Myoglobins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
    • C07K16/065—Purification, fragmentation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/25—Chemistry: analytical and immunological testing including sample preparation
    • Y10T436/25125—Digestion or removing interfering materials

Definitions

  • the present invention relates to a process for the separation of the main proteins from hemolyzed blood in order to allow the production on an industrial scale of proteins such as albumin, hemoglobin and gamma globulins.
  • the industrial-scale fractionation processes for plasma proteins or blood serum are based on selective precipitation techniques.
  • One of the well known methods is the Cohn method using selective ethanol precipitation.
  • Other methods use other chemical agents such as solvents, ammonium sulfate, caprylic acid, rivanol. These various processes have been applied mainly to obtaining and purifying albumin and gamma globulins.
  • hemoglobin elimination leads to the denaturation of this one. This.
  • the invention therefore proposes to remedy these drawbacks and to provide a process for the separation of the main proteins from hemolyzed blood, in undenatured form, capable of separating, with an extremely high degree of purity, the main proteins.
  • the main proteins such as albumin, hemoglobin, and if desired, y-globulins.
  • Another object of the invention is to provide such a method which makes it possible to economically treat extremely large quantities of hemolyzed blood, such as for example several tonnes or several tens of tonnes of placenta per day corresponding to several tens of thousands of liters of hemolyzed blood.
  • the subject of the invention is a process for the separation of the main proteins from hemolyzed blood, and in particular albumin and hemoglobin, in non-denatured form, in which a chromatographic separation step of albumin and hemoglobin, characterized in that a hemolyzed blood is subjected to a clarification step, then, preferably before any alcoholic precipitation of ⁇ -globulins, a chromatography of the hemolyzed blood is carried out on an anion exchange chromatographic support pH greater than 4.8 and less than 6.8, preferably less than 6 in the case of prior alcoholic precipitation of the y-globulins, the pH preferably being between 5 and 6, after which the hemoglobin leaving the chromatography column and the albumin obtained after elution.
  • the clarification is carried out at a pH of between 4 and 6, preferably between 4.8 and 5.4, in the presence of alcohol, preferably ethanol, at a concentration of less than 15% and preferably close 8%.
  • the hemolyzed blood being generally diluted, a concentration step is carried out, carried out on ultrafiltration systems having a cutoff threshold preferably of the order of 10,000 daltons, preferably so as to concentrate at least four times the hemolyzed blood. , as well as a diafiltration.
  • the chromatography step it is advantageous, after having obtained this hemoglobin by the chromatography step, to treat the filtrate by adding ethanol to about 25%, cold, with adjustment of the pH towards 7.
  • the immunoglobulin precipitate can then be recovered, for example by centrifugation or filtration, and then undergo any purification operations which may be necessary.
  • the corresponding supernatant or filtrate contains the hemoglobin in practically pure native form.
  • this hemoglobin is diluted with at least one volume of distilled water so as to lower the ethanol concentration sufficiently to avoid any subsequent denaturation.
  • the solution can be subjected to an ultrafiltration and diafiltration operation to recover the concentrated native hemoglobin ready to undergo other purification operations or other treatments necessary for its transformation into an artificial blood substitute.
  • the chromatography support is a support with very high mechanical properties, for example comprising a substrate made of silica beads.
  • the chromatography support is preferably the support called Spherodex e and described for example in the aforementioned French patent or in Luxembourg patent no 73094 filed on July 29, 1975, consisting of a porous silica coated with DEAE Dextran and prepared at the Institute Merieux.
  • 150 kg of human placentas are ground in the frozen state and dispersed in 150 liters of water, added with 22 liters of ethanol to reach a temperature of 0 ° C and an alcoholic concentration of approximately 8%.
  • the suspension thus obtained is stirred vigorously and then subjected to filtration on a 400-liter volume press equipped with a fabric of porosity 50 microns, to separate the blood juice from the placental tissues.
  • the recovered liquid occupies a volume of 270 liters.
  • Acetic acid or hydrochloric acid N is added indifferently, to adjust the pH to 5.10 with stirring at 0 ° C.
  • the suspension is injected at 3001 / h on a centrifuge. A neighboring precipitate of 7 to 10 kg depending on the day is eliminated.
  • the blood obtained in the supernatant is perfectly clear and occupies a volume of approximately 260 liters.
  • An ultrafilter equipped with a 10,000 dalton cutoff threshold membrane is used.
  • two spiral cartridges of 5 m 2 each, sold by Millipore, are well suited for this operation.
  • the ultrafiltrate removal rate is approximately 501 / h at a pressure of 4 bars at the inlet.
  • the total operation takes around 8 hours at + 4 ° C.
  • the final pH is around 5.25 and is not changed.
  • the ultrafilter can be reused more than 200 times without clogging.
  • the concentrated blood After standing for 15 hours at + 4 ° C., the concentrated blood contains a slight precipitate of euglobulins which is eliminated by centrifugation.
  • the pellet obtained represents a weight of 100 to 300 grams depending on the batch.
  • the clarified concentrated blood with a volume of 120 liters is then filtered without problems on a membrane with a porosity of 0.2 ⁇ and stored sterile pending the next step.
  • a column with a diameter of 16cm and a height of 1m is filled with 9 kg of Spherodex®. It is balanced in 0.01 M phosphate buffer pH 5.25, under sterile conditions already described (Tayot et al .: International Cooperation and blood derivatives, 1981, Editions Fondation Merieux, Lyon).
  • the 120 liters of concentrated and filtered blood obtained previously are injected into the column at a flow rate of 40 l / h, in a sterile manner through a filter with a porosity of 0.2 g.
  • the column is then rinsed with 40 liters of 0.01 M P0 4 buffer pH 5.25.
  • the 135-liter filtrate, containing hemoglobin and immunoglobulins, is completely free of albumin.
  • albumin fixed on the column with other proteins is then eluted by injection of 60 liters of a 20 g / l NaCl solution. A volume of 50 titles is sufficient to quantitatively recover albumin, or approximately 1200g g (8 g / kg placentas on average). This albumin solution must then be subjected to other known purification steps to be used in human therapy.
  • the preceding filtrate (135 liters) is adjusted to sodium chloride 8g / l, adjusted to pH 7, cooled to 0 ° C. 45 liters of ethanol cooled to -20 ° C are added gradually, with stirring and the temperature is finally adjusted to -5 ° C. After standing overnight, the suspension obtained is centrifuged at a flow rate of 300 l / h.
  • the precipitate of gamma globulin obtained weighs 900 grams on average over several batches, and must then be subjected to other purification operations already known, to prepare immunoglobulins which can be used in human therapy.
  • the supernatant obtained is perfectly clear. It contains hemoglobin practically free of immunoglobulins. It is diluted with 300 liters of water at 0 ° C to bring the alcoholic concentration below 10%. This solution is concentrated by ultrafiltration on the same module described above and can be diafiltered in the end to adjust the ionic strength and the composition of the solution according to its future use. The amount of hemoglobin thus obtained is 3700 g, or a yield close to 25 g / kg placentas.
  • Tests carried out at a pH of 5.0 show a reduction in the capacity of the column to retain albumin while at ph 5.2 albumin is completely retained.
  • Tests carried out at pH 6.5 have shown that if the albumin is fully retained, contamination of the support by the pigments of hemoglobin begins to appear.
  • the clarification step at pH 5.1 and 8% alcohol is carried out before and / or after the precipitation of the globulin fraction.

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  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
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  • Gastroenterology & Hepatology (AREA)
  • Zoology (AREA)
  • Toxicology (AREA)
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  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
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Abstract

The process comprises subjecting the hemolyzed blood to a clarification step at a pH of between 4 and 6 in the presence of less than 15% of alcohol, then, after concentration, effecting a chromatography by exchange of anions so as to separate the hemoglobin, the albumin being thereafter eluted. The hemoglobin and the globulins may then be separated out by precipitation with 25% alcohol at a pH of 7. The proteins obtained are in the native state.

Description

La présente invention a trait à un procédé de séparation des principales protéines du sang hémolysé afin de permettre l'obtention à l'échelle industrielle, des protéines telles que l'albumine, l'hémoglobine et les gamma-globulines.The present invention relates to a process for the separation of the main proteins from hemolyzed blood in order to allow the production on an industrial scale of proteins such as albumin, hemoglobin and gamma globulins.

Les procédés de fractionnement à l'échelle industrielle des protéines du plasma ou de sérum sanguin reposent sur les techniques de précipitation sélective. L'un des procédés, bien connu, est la méthode de Cohn utilisant la précipitation sélective par l'éthanol. D'autres procédés utilisent d'autres agents chimiques tels que des solvants, le sulfate d'ammonium, l'acide caprylique, le riva- nol. Ces différents procédés ont été appliqués principalement à l'obtention et à la purification de l'albumine et des gamma-globulines.The industrial-scale fractionation processes for plasma proteins or blood serum are based on selective precipitation techniques. One of the well known methods is the Cohn method using selective ethanol precipitation. Other methods use other chemical agents such as solvents, ammonium sulfate, caprylic acid, rivanol. These various processes have been applied mainly to obtaining and purifying albumin and gamma globulins.

Ils possèdent cependant un certain nombre d'inconvénients, parmi lesquels on peut citer une sélectivité limitée et donc l'obtention d'un degré du purification limité, des pertes en rendement par dénaturation en raison de la formation de polymères ou d'agrégats de protéines pendant les étapes de précipitation, et enfin des opérations industrielles fastidieuses, bruyantes et délicates comme les opérations de centrifugation, nécessitant un personnel habile et compétent.However, they have a certain number of drawbacks, among which there may be mentioned a limited selectivity and therefore the obtaining of a limited degree of purification, losses in yield by denaturation due to the formation of polymers or protein aggregates. during the precipitation stages, and finally tedious, noisy and delicate industrial operations such as centrifugation operations, requiring skillful and competent personnel.

En outre, dès lors que ces procédés sont mis en oeuvre sur du sang hémolysé, par exemple du sang placentaire qui constitue la source la plus importante de protéines sériques, les étapes connues d'élimination de l'hémoglobine conduisent à la dénaturation de celle-ci. Or, il serait souhaitable de disposer de sources importantes d'hémoglobine native en raison notamment de l'intérêt que peut présenter l'hémoglobine, comme source de sang artificiel.In addition, as soon as these methods are implemented on hemolyzed blood, for example placental blood which constitutes the most important source of serum proteins, the known stages of hemoglobin elimination lead to the denaturation of this one. this. However, it would be desirable to have significant sources of native hemoglobin due in particular to the advantage that hemoglobin can present, as a source of artificial blood.

On a donc déjà envisagé de réaliser des étapes de purification de protéines du plasma ou du sang hémolysé par chromatographie, et notamment par chromatographie d'échange d'ions, en utilisant des supports de chromatographie modernes tels que par exemple des microbilles de silice revêtues de DEAE Dextrane (voir par exemple le brevet français no 2319399 déposé le 29 juillet 1976.It has therefore already been envisaged to carry out purification steps for plasma proteins or hemolyzed blood by chromatography, and in particular by ion exchange chromatography, using modern chromatography supports such as for example silica microbeads coated with DEAE Dextrane (see for example French patent no 2319399 filed on July 29, 1976.

On a ainsi déjà envisagé de soumettre à une étape de séparation chromatographie le surnageant placentaire d'albumine obtenu après précipitation alcoolique des y-globulines, à pH 6,8 en présence de 25% d'éthanol, séparant ainsi l'hémoglobine de l'albumine, laquelle était ensuite récupérée par élution à l'aide d'une solution NaCI 1% (voir J. L.Tayot et al.; Chromatography of synthe- tic and biological polymers, 1978, Vol. 2, pages 95 à 109). Un procédé similaire a également été suggéré dans le brevet français précité dans lequel le surnageant de l'étape de précipitation alcoolique du bloc des globulines est soumis à une séparation chromatographique à un pH compris entre 6 et 7 après dilution dans de l'eau distillée pour diminuer la concentration en alcool, permettant ainsi de séparer l'albumine de l'hémoglobine qui traverse la colonne sans se fixer au support.It has thus already been envisaged to subject the placental albumin supernatant obtained after alcoholic precipitation of the y-globulins to a chromatography separation step, at pH 6.8 in the presence of 25% ethanol, thus separating the hemoglobin from the albumin, which was then recovered by elution using a 1% NaCl solution (see JLTayot et al .; Chromatography of synthesis and biological polymers, 1978, Vol. 2, pages 95-109). A similar process has also been suggested in the aforementioned French patent in which the supernatant of the alcoholic precipitation step of the globulin block is subjected to chromatographic separation at a pH of between 6 and 7 after dilution in distilled water to decrease the alcohol concentration, thus making it possible to separate the albumin from the hemoglobin which crosses the column without attaching to the support.

Cependant, malgré les avantages que l'on peut attendre de la séparation chromatographique, ces procédés de purification du sang hémolysé n'on pas pu être utilisés à l'échelle industrielle.However, despite the advantages that can be expected from chromatographic separation, these methods of purifying hemolyzed blood could not be used on an industrial scale.

Certes, on a déjà prévu de purifier l'albumine d'origine placentaire par des étapes de séparation chromatographique mais à condition d'éliminer tout d'abord l'essentiel de l'hémoglobine par une précipitation dénaturante, par exemple au chloroforme (voir J. L. Tayot et al.; Coopération internationale et dérivés sanguins-Talloires 1981; Ed. fondation Merieux), mais un tel procédé conduit à la dénaturation irréversible de l'hémoglobine. Par contre les opérations de séparation chromatographique précitées du surnageant d'origine placentaire contenant l'albumine et l'hémoglobine n'ont pas pu atteindre le stade industriel, notamment en raison d'une légère fixation d'hémoglobine sur le support, difficile à éluer complètement de sorte que l'accumulation des pigments, de cycle en cycle, sur le support entrave la capacité de fixation et la durée d'utilisation. De plus la solution injectée dans la colonne est très difficile à clarifier. Elle se trouble en continu, ce qui en rend la filtration très difficile. En outre elle contient des sels qui diminuent fortement la fixation de l'albumine.Admittedly, provision has already been made to purify albumin of placental origin by chromatographic separation steps, but on condition that the essential part of the hemoglobin is eliminated first by denaturing precipitation, for example with chloroform (see JL Tayot et al .; International cooperation and blood derivatives-Talloires 1981; Ed. Merieux foundation), but such a process leads to the irreversible denaturation of hemoglobin. On the other hand, the abovementioned chromatographic separation operations of the supernatant of placental origin containing albumin and hemoglobin could not reach the industrial stage, in particular due to a slight fixation of hemoglobin on the support, difficult to elute completely so that the accumulation of pigments, from cycle to cycle, on the support hinders the fixing capacity and the duration of use. In addition, the solution injected into the column is very difficult to clarify. It becomes cloudy continuously, which makes filtration very difficult. In addition it contains salts which strongly decrease the fixation of albumin.

L'invention se propose, en conséquence, de remédier à ces inconvénients et de fournir un procédé de séparation des principales protéines du sang hémolysé, sous forme non dénaturée, su- ceptible de séparer, avec un degré de pureté extrêmement élevé, les protéines principales telles que albumine, hémoglobine, et si on le désire, y-globulines.The invention therefore proposes to remedy these drawbacks and to provide a process for the separation of the main proteins from hemolyzed blood, in undenatured form, capable of separating, with an extremely high degree of purity, the main proteins. such as albumin, hemoglobin, and if desired, y-globulins.

Un autre objectif de l'invention est de fournir un tel procédé qui permette de traiter de façon économique des quantités extrêmement importantes de sang hémolysé, telles que par exemple plusieurs tonnes ou plusieurs dizaines de tonnes de placenta par jour correspondant à plusieurs dizaines de milliers de litres de sang hémolysé.Another object of the invention is to provide such a method which makes it possible to economically treat extremely large quantities of hemolyzed blood, such as for example several tonnes or several tens of tonnes of placenta per day corresponding to several tens of thousands of liters of hemolyzed blood.

L'invention a pour objet un procédé de séparation des principales protéines du sang hémolysé, et notamment de l'albumine et de l'hémoglobine, sous forme non dénaturée, dans lequel on effectue une étape de séparation chromatographique de l'albumine et de l'hémoglobine, caractérisé en ce que l'on soumet un sang hémolysé à une étape de clarification, puis, de préférence avant toute précipitation alcoolique de y-globulines, on effectue une chromatographie du sang hémolysé sur un support chromatographique échangeur d'anions à un pH supérieur à 4,8 et inférieur à 6,8, de préférence inférieur à 6 dans le cas d'une précipitation alcoolique préalable des y-globulines, le pH étant de préférence compris entre 5 et 6, après quoi on récupère séparément l'hémoglobine sortant de la colonne de chromatographie et l'albumine obtenue après élution.The subject of the invention is a process for the separation of the main proteins from hemolyzed blood, and in particular albumin and hemoglobin, in non-denatured form, in which a chromatographic separation step of albumin and hemoglobin, characterized in that a hemolyzed blood is subjected to a clarification step, then, preferably before any alcoholic precipitation of γ-globulins, a chromatography of the hemolyzed blood is carried out on an anion exchange chromatographic support pH greater than 4.8 and less than 6.8, preferably less than 6 in the case of prior alcoholic precipitation of the y-globulins, the pH preferably being between 5 and 6, after which the hemoglobin leaving the chromatography column and the albumin obtained after elution.

De façon particulièrement préférée, on réalise la clarification à un pH compris entre 4 et 6, de préférence entre 4,8 et 5,4, en présence d'alcool, de préférence éthanol, à une concentration inférieure à 15% et de préférence voisine de 8%.In a particularly preferred manner, the clarification is carried out at a pH of between 4 and 6, preferably between 4.8 and 5.4, in the presence of alcohol, preferably ethanol, at a concentration of less than 15% and preferably close 8%.

Le sang hémolysé étant en général dilué, on réalise une étape de concentration, conduite sur des systèmes d'ultrafiltration ayant un seuil de coupure de préférence de l'ordre de 10000 daltons, de préférence de façon à concentrer au moins quatre fois le sang hémolysé, ainsi qu'une diafiltration.The hemolyzed blood being generally diluted, a concentration step is carried out, carried out on ultrafiltration systems having a cutoff threshold preferably of the order of 10,000 daltons, preferably so as to concentrate at least four times the hemolyzed blood. , as well as a diafiltration.

Dans le cas, qui n'est pas préféré, dans lequel le sang hémolysé a subi préalablement des étapes de précipitation alcoolique, notamment pour la séparation des y-globulines, il est nécessaire d'effectuer une dilution dans de l'eau distillée ou un liquide analogue de façon que le taux d'alcool ne dépasse pas 15%. Ceci entraîne la nécessité de travailler sur des volumes très importants et présente d'autre part des inconvénients dus au fait que les additifs nécessaires dans le cas de la précipitation alcoolique des y-globulines diminuent les possibilités d'adsorption du support chromatographique.In the case, which is not preferred, in which the hemolyzed blood has previously undergone alcoholic precipitation steps, in particular for the separation of γ-globulins, it is necessary to carry out a dilution in distilled water or a analogous liquid so that the alcohol level does not exceed 15%. This leads to the need to work on very large volumes and on the other hand has drawbacks due to the fact that the additives necessary in the case of the alcoholic precipitation of the y-globulins reduce the possibilities of adsorption of the chromatographic support.

Si l'on souhaite séparer les globulines et notamment y-globulines de l'hémoglobine purifiée, débarrassée de l'albumine, on peut avantageusement, après avoir obtenu cette hémoglobine par l'étape de chromatographie, traiter le filtrat par addition d'éthanol à environ 25%, à froid, avec ajustement du pH vers 7. Le précipité d'immunoglobulines peut alors être récupéré par exemple par centrifugation ou filtration et subir ensuite les opérations de purification éventuellement nécessaires. Le surnageant ou filtrat correspondant contient l'hémoglobine sous forme native pratiquement pure. De préférence, cette hémoglobine est diluée avec au moins un volume d'eau distillée de façon à abaisser la concentration en éthanol suffisamment pour éviter toute dénaturation ultérieure. La solution peut être soumise à une opération d'ultrafiltration et de diafiltration pour récupérer l'hémoglobine native concentrée prête à subir d'autres opérations de purification ou autres traitements nécessaires à sa transformation en substitut artificiel du sang.If it is desired to separate the globulins and in particular γ-globulins from the purified hemoglobin, free of albumin, it is advantageous, after having obtained this hemoglobin by the chromatography step, to treat the filtrate by adding ethanol to about 25%, cold, with adjustment of the pH towards 7. The immunoglobulin precipitate can then be recovered, for example by centrifugation or filtration, and then undergo any purification operations which may be necessary. The corresponding supernatant or filtrate contains the hemoglobin in practically pure native form. Preferably, this hemoglobin is diluted with at least one volume of distilled water so as to lower the ethanol concentration sufficiently to avoid any subsequent denaturation. The solution can be subjected to an ultrafiltration and diafiltration operation to recover the concentrated native hemoglobin ready to undergo other purification operations or other treatments necessary for its transformation into an artificial blood substitute.

Conformément à l'invention, le support de chromatographie est un support à très hautes propriétés mécaniques, comprenant par exemple un substrat en billes de silice.According to the invention, the chromatography support is a support with very high mechanical properties, for example comprising a substrate made of silica beads.

Le support de chromatographie est de préférence le support dénommé Sphérodexe et décrit par exemple dans le brevet français précité ou dans le brevet luxembourgeois no 73094 déposé le 29 juillet 1975, constitué d'une silice poreuse revêtue de DEAE Dextran et préparée à l'Institut Merieux.The chromatography support is preferably the support called Spherodex e and described for example in the aforementioned French patent or in Luxembourg patent no 73094 filed on July 29, 1975, consisting of a porous silica coated with DEAE Dextran and prepared at the Institute Merieux.

D'autres avantages et caractéristiques de l'invention apparaîtront à la lecture de la description suivante, faite à titre d'exemple non limitatif.Other advantages and characteristics of the invention will appear on reading the following description, given by way of nonlimiting example.

1) Etape de clarification du sang placentaire hémolysé1) Clarification stage of hemolyzed placental blood

150 kg de placentas humains sont broyés à l'état congelé et dispersés dans 150 litres d'eau, additionnés de 22 litres d'éthanol pour atteindre une température de 0°C et une concentration alcoolique d'environ 8%. La suspension ainsi obtenue est agitée vigoureusement puis soumise à une filtration sur pressoir de volume 400 litres équipé d'une toile de porosité 50 microns, pour séparer le jus sanguin des tissus placentaires. Le liquide récupéré occupe un volume de 270 litres. Il est additionné d'acide acétique ou d'acide chlorhydrique N indifféremment, pour ajuster le pH à 5,10 sous agitation à 0°C. Après 2 heures d'attente, la suspension est injectée à 3001/h sur centrifugeuse. Un précipité voisin de 7 à 10 kg suivant les jours est éliminé. Le sang obtenu dans le surnageant est parfaitement limpide et occupe un volume de 260 litres environ.150 kg of human placentas are ground in the frozen state and dispersed in 150 liters of water, added with 22 liters of ethanol to reach a temperature of 0 ° C and an alcoholic concentration of approximately 8%. The suspension thus obtained is stirred vigorously and then subjected to filtration on a 400-liter volume press equipped with a fabric of porosity 50 microns, to separate the blood juice from the placental tissues. The recovered liquid occupies a volume of 270 liters. Acetic acid or hydrochloric acid N is added indifferently, to adjust the pH to 5.10 with stirring at 0 ° C. After 2 hours of waiting, the suspension is injected at 3001 / h on a centrifuge. A neighboring precipitate of 7 to 10 kg depending on the day is eliminated. The blood obtained in the supernatant is perfectly clear and occupies a volume of approximately 260 liters.

2) Etape de concentration et diafiltration du sang clarifié2) Concentration and diafiltration stage of clarified blood

Un ultrafiltre équipé d'une membrane de seuil de coupure 10000 daltons est utilisé. Par exemple deux cartouches spirales de 5 m2 chacune, commercialisées par Millipore, conviennent bien pour cette opération. Après concentration à environ 60 litres, le pH est ajusté à 5,50 avec de la soude N et on diafiltre ensuite à volume constant avec environ 200 litres d'eau déminéralisée. Le débit d'élimination de l'ultrafiltrat est d'environ 501/h pour une pression de 4 bars à l'entrée. L'opération totale dure environ 8 heures à +4°C. Le pH final est voisin de 5,25 et n'est pas modifié. Après régénération l'ultrafiltre peut être ainsi réutilisé plus de 200 fois sans colmatage.An ultrafilter equipped with a 10,000 dalton cutoff threshold membrane is used. For example, two spiral cartridges of 5 m 2 each, sold by Millipore, are well suited for this operation. After concentration to approximately 60 liters, the pH is adjusted to 5.50 with sodium hydroxide and then diafiltered to constant volume with approximately 200 liters of demineralized water. The ultrafiltrate removal rate is approximately 501 / h at a pressure of 4 bars at the inlet. The total operation takes around 8 hours at + 4 ° C. The final pH is around 5.25 and is not changed. After regeneration, the ultrafilter can be reused more than 200 times without clogging.

Après repos de 15 heures à +4°C, le sang concentré contient un léger précipité d'euglobulines qui est éliminé par centrifugation. Le culot obtenu représente un poids de 100 à 300 grammes selon les lots. Le sang concentré clarifié d'un volume de 120 litres est ensuite filtré sans problèmes sur une membrane de porosité 0,2µ et conservé stérilement dans l'attente de l'étape suivante.After standing for 15 hours at + 4 ° C., the concentrated blood contains a slight precipitate of euglobulins which is eliminated by centrifugation. The pellet obtained represents a weight of 100 to 300 grams depending on the batch. The clarified concentrated blood with a volume of 120 liters is then filtered without problems on a membrane with a porosity of 0.2 μ and stored sterile pending the next step.

3) Etape de séparation chromatographique3) Chromatographic separation step

Une colonne de diamètre 16cm et de hauteur 1 m est remplie de 9 kg de Sphérodex®. Elle est équilibrée en tampon phosphate 0,01 M pH 5,25, dans des conditions stériles déjà décrites (Tayot et coll.: Coopération Internationale et dérivés sanguins, 1981, Editions Fondation Merieux, Lyon). Les 120 litres de sang concentré et filtré obtenus précédemment sont injectés dans la colonne à un débit de 40 1/h, de manière stérile à travers un filtre de porosité 0,2 g. La colonne est ensuite rincée par 40 litres de tampon P04 0,01 M pH 5,25. Le filtrat d'un volume de 135 litres, contenant l'hémoglobine et les immunoglobulines, est totalement débarrassé de l'albumine. Il est conservé à +2°C en attendant l'étape suivante. L'albumine fixée sur la colonne avec d'autres protéines est ensuite éluée par injection de 60 litres d'une solution de NaCI 20 g/1. Un volume de 50 titres suffit pour récupérer quantitativement l'albumine, soit environ 1200g g (8 g/kg placentas en moyenne). Cette solution d'albumine doit ensuite être soumise à d'autres étapes connues de purification pour être utilisable en thérapeutique humaine.A column with a diameter of 16cm and a height of 1m is filled with 9 kg of Spherodex®. It is balanced in 0.01 M phosphate buffer pH 5.25, under sterile conditions already described (Tayot et al .: International Cooperation and blood derivatives, 1981, Editions Fondation Merieux, Lyon). The 120 liters of concentrated and filtered blood obtained previously are injected into the column at a flow rate of 40 l / h, in a sterile manner through a filter with a porosity of 0.2 g. The column is then rinsed with 40 liters of 0.01 M P0 4 buffer pH 5.25. The 135-liter filtrate, containing hemoglobin and immunoglobulins, is completely free of albumin. It is stored at + 2 ° C pending the next step. The albumin fixed on the column with other proteins is then eluted by injection of 60 liters of a 20 g / l NaCl solution. A volume of 50 titles is sufficient to quantitatively recover albumin, or approximately 1200g g (8 g / kg placentas on average). This albumin solution must then be subjected to other known purification steps to be used in human therapy.

La colonne après lavage par une solution d'HCI 0,1 N d'alcool est ensuite prête pour un nouveau cycle d'utilisation.The column after washing with a 0.1 N HCl solution of alcohol is then ready for a new cycle of use.

Plus de 50 cycles ont été effectués sur la même colonne, sans perte d'efficacité.More than 50 cycles were carried out on the same column, without loss of efficiency.

4) Séparation finale des gamma-globulines et de l'hémoglobine4) Final separation of gamma globulins and hemoglobin

Le filtrat précédent (135 litres) est ajusté en chlorure de sodium 8g/l, ajusté à pH 7, refroidi à 0°C. 45litres d'éthanol refroidi à -20°C sont ajoutés progressivement, sous agitation et la température est ajustée à -5°C en final. Après un repos d'une nuit, la suspension obtenue est centrifugée à un débit de 300 1/h.The preceding filtrate (135 liters) is adjusted to sodium chloride 8g / l, adjusted to pH 7, cooled to 0 ° C. 45 liters of ethanol cooled to -20 ° C are added gradually, with stirring and the temperature is finally adjusted to -5 ° C. After standing overnight, the suspension obtained is centrifuged at a flow rate of 300 l / h.

Le précipité de gammaglobulines obtenu pèse 900 grammes en moyenne sur plusieurs lots, et doit être ensuite soumis à d'autres opérations de purification déjà connues, pour préparer des immunoglobulines utilisables en thérapeutique humaine.The precipitate of gamma globulin obtained weighs 900 grams on average over several batches, and must then be subjected to other purification operations already known, to prepare immunoglobulins which can be used in human therapy.

Le surnageant obtenu est parfaitement limpide. Il contient l'hémoglobine pratiquement débarrassée d'immunoglobulines. Il est dilué avec 300 litres d'eau à 0°C pour faire descendre la concentration alcoolique en dessous de 10%. Cette solution est concentrée par ultrafiltration sur le même module décrite précédemment et peut être diafiltrée en final pour ajuster la force ionique et la composition de la solution en fonction de son utilisation future. La quantité d'hémoglobine ainsi obtenue est de 3700 g, soit un rendement voisin de 25 g/kg placentas.The supernatant obtained is perfectly clear. It contains hemoglobin practically free of immunoglobulins. It is diluted with 300 liters of water at 0 ° C to bring the alcoholic concentration below 10%. This solution is concentrated by ultrafiltration on the same module described above and can be diafiltered in the end to adjust the ionic strength and the composition of the solution according to its future use. The amount of hemoglobin thus obtained is 3700 g, or a yield close to 25 g / kg placentas.

Des essais effectués à un pH de 5,0 montrent une diminution de la capacité de la colonne à retenir l'albumine alors qu'au ph 5,2 l'albumine est totalement retenue. Des essais effectués à pH 6,5 ont montré que si l'albumine est entièrement retenue, une contamination du support par les pigments de l'hémoglobine commence à apparaître.Tests carried out at a pH of 5.0 show a reduction in the capacity of the column to retain albumin while at ph 5.2 albumin is completely retained. Tests carried out at pH 6.5 have shown that if the albumin is fully retained, contamination of the support by the pigments of hemoglobin begins to appear.

Dans le cas, non préféré, dans lequel on soumet le sang hémolysé à une précipitation alcoolique à pH 7 pour séparer les y-globulines, l'étape de clarification à pH 5,1 et 8% d'alcool est conduite avant et/ou après la précipitation de la fraction globuli- nique.In the case, not preferred, in which the hemolyzed blood is subjected to an alcoholic precipitation at pH 7 to separate the y-globulins, the clarification step at pH 5.1 and 8% alcohol is carried out before and / or after the precipitation of the globulin fraction.

Claims (12)

1. A process for separating the principal proteins from haemolyzed blood, and in particular the albumin and the haemoglobin in the non-denatured form, in which a chromatographic separation of the albumin and the haemoglobin is effected, characterized in that a haemolyzed blood is subjected to a clarification step, then a chromatography of the haemolyzed blood is effected on an anion exchanging chromatographic support at a pH higher than 4.8 and lower than 6.8, after which the haemoglobin issuing from the chromatographic column and the albumin obtained after elution are recovered separately.
2. A process according to claim 1, characterized in that the chromatography is effected at a pH of between 5 and 6.
3. A process according to claim 1 or 2, characterized in that the clarification step is effected at a pH between 4 and 6 in the presence of alcohol at a concentration lower than 15%.
4. A process according to claim 3, characterized in that the clarification is effected at a pH of between 4.8 and 5.4.
5. A process according to claim 3 or 4, characterized in that the clarification step is effected in the presence of alcohol at a concentration of around 8%.
6. A process according to any one of claims 3 to 5, characterized in that the clarification step is effected in the presence of ethanol.
7. A process according to any one of claims 1 to 6, characterized in that the chromatography is effected before any separation of the y globulins.
8. A process according to claim 7, characterized in that the y globulins are separated from the purified haemoglobin.
9. A process according to claim 8, characterized in that the y globulins are separated by precipitation with alcohol at about 25% and with a pH in the region of 7.
10. A process according to any one of claims 1 to 9, characterized in that a support of porous silica coated with DEAE dextran is employed for the chromatography.
11. A process according to any one of claims 1 to 10, characterized in that the chromatographic column is equilibrated with an 0.01 M phosphate buffer, then is rinsed in the same buffer, the albumin being eluted with a 20g/I solution of NaCI.
12. A process according to any one of claims 1 to 11, characterized in that a concentration of a diluted haemolyzed blood, in particular by ultrafiltration and diafiltration, is effected before the chromatography.
EP84401303A 1983-07-07 1984-06-21 Process for the preparation of the principal proteins of hemolysed blood in a non denatured form Expired EP0132178B1 (en)

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AT84401303T ATE30844T1 (en) 1983-07-07 1984-06-21 PROCESS FOR THE PRODUCTION OF THE MAJOR PROTEINS OF HAEMOLYZED BLOOD IN AN NON-DENATURED FORM.

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FR8311323 1983-07-07
FR8311323A FR2548670B1 (en) 1983-07-07 1983-07-07 PROCESS FOR THE PREPARATION OF THE MAIN BLOOD HEMOLYSIS PROTEINS IN UNDENATURED FORM

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EP0132178A1 EP0132178A1 (en) 1985-01-23
EP0132178B1 true EP0132178B1 (en) 1987-11-19

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JP (1) JPS60149526A (en)
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US4810391A (en) * 1987-11-06 1989-03-07 Bio-Rad Laboratories, Inc. Separation of hemoglobin A2 from hemoglobin mixture
US4980058A (en) * 1987-11-06 1990-12-25 Bio-Rad Laboratories, Inc. Separation of hemoglobin A2 from hemoglobin mixture
US5277818A (en) * 1988-10-31 1994-01-11 The Green Cross Corporation Albumin preparation and process for preparing the same
US5250662A (en) * 1989-10-05 1993-10-05 Alpha Therapeutic Corporation Albumin purification
CA2022165C (en) * 1989-10-05 1999-11-23 Chong E. Chang Albumin purification
US5650388A (en) * 1989-11-22 1997-07-22 Enzon, Inc. Fractionated polyalkylene oxide-conjugated hemoglobin solutions
US5250663A (en) * 1990-04-19 1993-10-05 Miles Inc. Preparing essentially monomeric normal human serum albumin
US5264555A (en) * 1992-07-14 1993-11-23 Enzon, Inc. Process for hemoglobin extraction and purification
US5840851A (en) * 1993-07-23 1998-11-24 Plomer; J. Jeffrey Purification of hemoglobin
CA2106612C (en) * 1993-09-21 2001-02-06 Diana Pliura Displacement chromatography process
US5545328A (en) * 1993-09-21 1996-08-13 Hemosol Inc. Purification of hemoglobin by displacement chromatography
US5631219A (en) * 1994-03-08 1997-05-20 Somatogen, Inc. Method of stimulating hematopoiesis with hemoglobin
US6242417B1 (en) 1994-03-08 2001-06-05 Somatogen, Inc. Stabilized compositions containing hemoglobin
US5741894A (en) * 1995-09-22 1998-04-21 Baxter International, Inc. Preparation of pharmaceutical grade hemoglobins by heat treatment in partially oxygenated form
JP2002517406A (en) * 1998-06-01 2002-06-18 ジェネンテック・インコーポレーテッド Separation of protein monomers from aggregates by using ion exchange chromatography

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US3657116A (en) * 1971-05-05 1972-04-18 Wolfgang Haller Process for the separation of blood components
LU73094A1 (en) * 1975-07-29 1977-03-24
US4100149A (en) * 1975-08-28 1978-07-11 Rhone-Poulenc Industries Method of separating proteins by ion exchange
FR2321932A1 (en) * 1975-08-28 1977-03-25 Rhone Poulenc Ind Separating proteins from aq. soln. contg. industrial effluent - by passing through ion exchange resin on mineral support
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DE2718326A1 (en) * 1977-04-25 1978-10-26 Behringwerke Ag NEW PROTEIN AND METHOD FOR THE PRODUCTION THEREOF

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EP0132178A1 (en) 1985-01-23
JPS60149526A (en) 1985-08-07
ES8504837A1 (en) 1985-04-16
CA1234047A (en) 1988-03-15
JPH0533239B2 (en) 1993-05-19
ES534008A0 (en) 1985-04-16
FR2548670A1 (en) 1985-01-11
DE3467504D1 (en) 1987-12-23
FR2548670B1 (en) 1985-10-25
US4764279A (en) 1988-08-16
SU1590031A3 (en) 1990-08-30
ATE30844T1 (en) 1987-12-15

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